This project describes the role of the lab of Stephanie London in the Laboratory of Respiratory Biology in support of her epidemiologic studies. The laboratory is engaged in analysis of samples from Dr. London's epidemiologic studies of respiratory disease. In the past year, we have focused on the study of childhood asthma in Mexico City (described under project entitled Genetic And Environmental Factors In Childhood Respiratory Health). The laboratory uses PCR technology to identify genetic polymorphisms in these samples. The methods used include RFLP-PCR, ARMS-PCR and real-time PCR (TaqMan). We also use the DNA sequencing core for two purposes. The first is identifying whether polymorphisms which have not been characterized with respect to their frequency are common enough in our Mexican population to warrant our consideration. We do this on a set of 20 quality control samples. The other use of the sequencing core is to generate controls of known genotype for whichever genotyping method that we are using. This year we have focused on the case-parent triad study of childhood asthma in Mexico City. Data collection is nearly complete in this study and we will have 500 case parent triads plus siblings on two-thirds of triads. We also have about 100 incomplete triads, missing the father. We have been conducting genetic analyses rather than waiting for the study to complete. Our Mexico City population is unique in being exposed to the highest ozone levels in North America. Although ozone has long been known to exacerbate asthma, evidence for a role of ozone in incidence of asthma is increasing. Linkage studies in mice indicate that aspects of pulmonary response to ozone that are relevant to asthma pathogenesis are under genetic control. Thus the ability to examine genetic susceptibility may help clarify the role on ozone in asthma and respiratory impairment. Because of the high exposure of our Mexico City population to ozone, we have primarily concentrated on genes that are both plausibly involved in asthma pathogenesis and known or suspected to be involved in pathways or response to ozone. These genes are predominantly involved in response to oxidative stress and inflammation. DNA extraction is done by the contract laboratory Bioserve. This is a labor intensive process in part because of the database processing and verification. In the past year, we have shifted our focus from individual SNPs to haplotype analysis. This entails greater genotyping requirement for each gene. To gain this expertise, we have been attending a journal club on haplotype analysis at UNC and also used lab funds to send the technician, Huiling Li, to a course on SNPs selection and haplotype analysis at University of Washington. In the next fiscal year, we will also be beginning a collaboration with the SAPALDIA study which involves the genotyping of 5,000 samples from subjects in a very well characterized air pollution cohort. We will be looking at genes that may modify respiratory and cardiovascular effects of air pollution.